Process for the production of digitoxigenin and intermediates



Patented Jan. 17, 1961 CH: CH: 2,968,596 onion CHzOAc PROCESS FOR THEPRODUCTION OF DIGITOXI- 5:9 (5:0

GENIN AND INTERMEDIATES CH1 5 Peter D. Meister, Kalamazoo Township,Kalamazoo A County, and Herbert C. Murray, Hickory Corners, Mich.,assignors to The Upjohn Company, Kalamazoo, Mich., a corporation ofMichigan No Drawing. Original application June 7, 1955, Ser.

No. 513,900. Divided and this application Nov. 17, 1958, Ser. No.774,117

a? as 4 Claims. Cl. 195-51 The present invention relates to steroids andis more l particularly concerned with a process of production for CH3CH3 digitoxigenin and the new intermediates of this process: CHzOR CHR4,14-pregnadiene-3,20-dione; 3a,2l-dihydroxyand 21- acyloxy-3a-hydroxy-14-pregnen-20-ones; 21-acyloxy-3u- 20 0H; CH3 I(-arylsulfonyloxy) 14-pregnen-20-ones; 3;3,21-dihydroxyand313,2l-diacyloxy-14-pregnen-20-ones; 3,B,15[3,21-trihydroxyand313,2l-diacyloxyJSfl-hydroxy-14a-halopregnan-20-ones;35-acyloxy-ISB-hydroxy-14a-halo-14a-card- 0H 20(22)-en0lide and3,6-hydroxyand 3 8-acyloXy-l4(l5)- 25 BI oxido-20(22)-cardenolide. R0A00 This invention is .a continuation-in-part of application SerialNuinber'450,526, filed August 17, 1954,.now U.S. (VIIIa) R 11 (vua) R- HPatent No. 2,889,255, and a division of application Serial (Vmb) No.513,900, filed June 7, 1955. l

The process and compounds of the present invention are illustrativelypresented by the following formulae CH3 and equations: CH-CO cH-co 0 C vH: CH8 CH2 CH:/\ \CHZ- =0 =0 l CH: CH:

E H o 40 i A00 R0 OAc (IX) (Xv R=H 0: 0: (Kb) l =Ac I II I! OHOphiobolus a herpotrz'chus v CH3 CH: CH3 CH2 011101: 3111012 =0 C=O CH:CH:

H,(Pd) (XIa) R=H O 0 (XIb) R=Ac wherein AcO represents an acyloxy group,Ac being the acyl radical of an organic carboxylic acid containing fromone to eight carbon atoms inclusive and TsO is an arylsulfonyloxy group.In the preferred embodiment of the l 6 process the acyl radical Ac is ofa hydrocarbon carboxylic acid containing from one to eight carbon atomsand the (P711) R=H (IIIa) R=H R=Ac (IIIb) R=Ac arylsulfonyloxy group TsOis a para-toluenesulfonyloxy group.

The process of the present invention compirses: pyrolysis of a15ot-acyloxyprogesterone (I) or alternatively dehydration of a14a-hydroxyprogesterone to yield 4,14- pregnadiene-3,20-dione (II);subjecting 4,14-pregnadiene- 3,20dine (II) to fermentation by Ophiobolusherpotricims to obtain 21-hydroxy-4,l4-pregnadiene-3,20-dione (III)which by hydrogenation with hydrogen and a palladium-catalyst in a basicmedium yields 2l-hydroxy-14- pregnene-3,20-dione (IVa); esterificationof (PM) by conventional means to obtain 2l-acyloxy-l4-pregnene- 3,20dione (IVb); reducing the thus-obtained acyloxy-l4- pregnene-3,20 dione(IVb) with sodium borohydride to give 21 acyloxy 30chydroxy-l4-pregnen-20-one (Vb) which by esterification with anarylsulfonyl chloride yields 21acyloxy-3warylsulfonyloxy-l4-pregnen-20-one (VIb); converting the3tt-ester (VIb) to a Zip-ester by reacting compound (VIb) with ahydrocarbon carboxylic acid containing from one to eight carbon atoms,inclusive, in the presence of an alkali salt of the same hydrocarboncarboxylic acid to give 318,21-diacyloxy-l4-pregnen-20-one (VIIb);treating 35,2l-diacyloxy-l4-pregnen- 20-one with a hypobromous acid(HBrO) preferably N-bromoacylamide in the presence of an acid to yield3,8,21 diacyloxy-lSB-hydroxy-l4ot-bromopregnan-20-one (VIIIb) which bysubmitting to a Reformatsky reaction (ethyl-bromoacetate and zinc)yielded 3B-acyloxy-15phydroxy-14a-bromo-l4a-card-20(22)-enolide (IX);heating compound IX with a mild base, such as sodium or potassiumacetate in an organic polar solvent such as acetone, dioxane, tertiarybutyl alcohol to yield 3fi-acyloxy-14(l5)-oxido-20-(22)-cardenolide(Xb); hydrolyzing the 3,8 acyloxy-14(l5)oxido-20(22)-cardenolide (Xb)thru fermentation with Rhizopus shanghaiensis to yield3,B-'hydroxy-14(l5)-oxido-20(22)-cardenolide (Xa) and treating compound(Xa) dissolved in an o'rganic solvent, for example, dioxane with analkali metal borohydride to reduce the 14(15)-oxido bond and thus togive 3p,14fi-dihydroxy-20(22)-cardenolide (digitoxigenin) (Xla).

It is an obiect of the present invention to provide a process for thepreparation of digitoxigenin. Another object of the instant invention isto provide 4,14-pregnadiene-3,20-dione; 21-hydroxyand 21-acyloxy-4,l4-pregnadiene-3,20-dione; 3a,21-dihydroxyand 2l-acyloxy-3u-hydroxy-4,14-pregnadien-20-ones; 2l-acyloxy-3a-(arylsulfonyloxy) 14pregnen 2O ones; 313,21 dihydroxyand 318,21 diacyloxy 14 pregnen 20ones; 3B,15,21-trihydroxyand 35,21-diacyioxy-15,8-hydroxy- 14ozbromopregnan 20 ones; 35,155 dihydroxyand 35 acyloxy 155 hydroxy 14ccbromopregnan-ZO-ones; 3,8.15fl-dihydroxyand 3,8-acyloxy-15fl-hydroxy14cc bromo 14oz card (22) enolide and 3fl-hydroxyand3,8-acy'oxy-l4(l5)-oxido-20(22)- cardenolide. It is another object ofthe present invention to provide a process for the preparation of thesecompounds. Other objects will be apparent to those skilled in the art towhich this invention pertains.

The instant novel process is a valuable new synthesis for puredigitoxigenin from readily available 14- or 15-oxygenated progesterone,available from progesterone. Digitoxigenin is theaglycon of the naturaloccurringe digitoxin and possesses almost equal cardiac activity.Furthermore digitoxigenin can be converted (Chen, 1943, E'derfield etal. ll. Am; Chem. Soc. 69, 2235 (1947), into the synthetic glycoside,glucose-digitoxigenin, in which form, according to Chen et al., J.Pharmacol. Exptl. Therap. 103, 420 (1951), it possesses almost threetimes the cardiac activity of digitoxin. Synthetic preparation of a purecardiac glycoside or its aglycon has the specific advantage of providingsimplified dosage 'by Weight instead of dosage based upon bioassayagainst primary standards as it is .now necessary with the variousdigitalis preparations. 1 i

The starting materials of the present process are14ahydroxyprogesterone, prepared as shown, for example, in US. Patent2,670,358, and l5a-hydroxyprogesterone esters, preferredly thel5a-acyloxy esters wherein the acyloxy group is AcO, Ac being the acylradical of a carboxylic acid containing from one to eight carbon atoms,inclusive, the preparations of which are shown in Preparations 1 through4.

In carrying out the process of the instant invention the selected15a-hydroxyprogesterone-lSa-acylate (I) is pyrolyzed suitably in acombustion tube in a combustion furnace at a temperature between 275 and400 degrees centigrade. In the preferred embodiment of the invention thepyrolysis of the selected 15u-acyloxyprogesterone (I) to obtain4,l4-pregnadiene-3,2O dione (II) is carried out by distilling compound(1) thru a tube in an inert gas atmosphere, extracting the distillatewith suitable organic water-immiscible solvents such as methylenedichloride, ethylene dichloride, chloroform, carbon tetrachloride,Skellysolve B hexane hydrocarbons, and the like and purifying the thusobtained material by conventional means such as recrystallization,extraction or chromatography or combinations thereof. Alternatively,14a-hydroxyprogesterone may be used as starting material. Thel4a-hydroxyprogesterone is dehydrated by heating it, dissolved in anacid anhydride, such as acetic anhydride in the presence of an equal, ortwo to three times larger Weight of potassium acid sulfate compared tothe weight of the steroid. Such a reaction mixture is convenientlyheated on the steam bath and after a period of half an hour, cooled withice, diluted with water and maintained at room temperature for sixteenhours. From the aqueous solution the 4,14-pregnadiene-3,20-dione may beisolated by extraction and purified by conventional means such aschromatography, extractions and recrystallization or combinationsthereof. The thus-obtained 4,14-pregnadiene-3,20 dione (II) is submittedto fermentation by Ophiobolus herpotrichus in a suitable fermentationmedium containing assimilable carbohydrates and nitrogen such asproteins or amino acids, and mineral factors, as shown in the example.The fermentation process is aided by aeration and agitation as well asby regulating the temperature to about 25 to 32 degrees centigrade. Inthe preferred embodiment of the invention, Ophiobolus herpotrichus isfirst grown in the fermentation medium under aeration and agitation fora period of 24 hours before the additon of the steroid to be fermented.After a period of twelve to 72 hours the fermentation is interrupted andthe steroids in the mixture are isolated by conventional means such asextraction with organic solvents, for example, methylene dichloride,ethylene dichloride, chloroform, acetone, and mixtures thereof. From theextractives the 2l-hydroxy- 4,l4-pregnadiene-3,20-dione (Illa) isobtained by conventional means such as trituration of the oily residuesafter the extraction solvent has been removed, recrystallization,chromatography or combination of such means.

21-hydroxy-4,14-pregnadiene-3,20-dione (IIIa), thus prepared, isthereupon hydrogenated, suitably dissolved in an organic solvent such asmethanol, ethanol, acetone, dioxane, tertiary butyl alcohol, withmethanol preferred and in the presence of palladium in a basic mediumsuch as, for example, palladium on a zinc oxide, cadmium oxide, zinccarbonate or cadmium carbonate carrier, or palladium in'potassiumhydroxide, sodium hydroxide, barium hydroxide solution, and the like.The thus ob tained 2l-hydroxy-14-pregnene 3,20-dione (IVa) is isolatedfrom the reaction mixture by eliminating by filtration or selectiveextraction of the steroid the catalyst material, evaporating theresulting solution, and extracting. 'recrystallizing or chromatographingthe remaining residue to obtain pure 21-hydroxy-l4-pregnene-3,20- dione(IVa).

Instead of hvdrogenating 2l-hydroxy-4,14-pregnadiene- 3,20-dione (Illa)the 21-acyloxy-4,14-p1'egnadiene'3,20-

aeeasee dione (IIIb) may be hydrogenated to give the corresponding21-acyloxy-14-pregnene-3,20-dione (IVb). 21-acy1-oxy-14-pregnene-3,20-dione may also be obtained by esterification of the21-hydroxy-14-pregnene-3,20-dione (IVa). Esterification of either the21-hydroxy-4,14- pregnadiene-3,20dione (Ilia) or of 21-hydroxy-14-pregnene-3,20-dione (IVa) is carried out by conventional means such asby mixing either of the starting compounds with an acylating agent, forexample, ketene, a ketene of a selected acid, an acid, such or formicacid, an acid chloride or acid bromide, an acid anhydride or other.known acylating agents, usually in a solvent such as, for example,pyridine or the like or an inert solvent, including solvents likebenzene, toluene, ether, and the like, and heating to a temperaturebetween about zero degrees centigrade and the boiling point of thereaction mixture, usually about room temperature (twenty to thirtydegrees centigrade) for a period between about a half an hour and about96 hours. The time of reaction is somewhat dependent upon thetemperature at which the reaction is carried out, the acylating agent,and the ratio of reactants. The reaction mixture is quenched with ice orcold water and the product is collected in an organic solvent which isthereafter washed with successive portions of a mildly basic solutionand water to obtain a solution of the product which is essentiallyneutral. In some instances the product recrystallized from the reactionmixture, in which case it may be ad vantageous to separate the productby filtration or other means, wash with water and thereafter purify byconventional means such as for example by recrystallization from asuitable solvent or chromatographic purification, as deemed necessary.The acyloxy groups preferably replacing the 21-hydroxyl groups in either21-hydroxy- 4,l4-pregnadiene-3,20-dione or 2l-hydroxy-l4-pregnene-3,20-dione are, the acyloxy radicals of hydrocarbon carboxylic acidcontaining from one to eight carbon atoms, inclusive. Obviously othercarboxylic acids can be used, however they are of no special advantagesince these acyloxy residues are eventually removed in the subsequentand final steps of the present synthesis, and since such carboxylic acidgroups require the use of more expensive acids than those of the commonhydrocarbon carboxylic acids containing from one to eight carbon atoms,inclusive, such as formic, acetic, propionic, butyric, isobutyric,valeric, isovaleric, hexanoic, heptanoic, octanoic,,B-cyclopentylpropionic, benzoic, toluic, phenylacetic, and the like.

The thus obtained 21-hydroxy-14-pregnene-3,20-dione (IVa) or in thepreferred embodiment the thus obtained 2l-acyloxy-14-pregnene-3,20-dione(IVb) is then reduced with sodium borohydride to give the corresponding21- hydroxyor 21-acyloxy-3a-hydroxy-14-prcgnen-20-one. The reduction ispreferably carried out while the selected steroid is dissolved indioxane, ether or tetrahydrofuran at a temperature of minus ten to plus20 degrees. The sodium borohydride is preferably added under vigorousstirring dissolved in a very small amount of sodium or potassiumhydroxide solution. A concentration of 50 to 100 milligrams of sodiumborohydride per milliliter of 0.05 to 0.2 normal sodium hydroxidesolution is used. The reaction time is between five to ten minutes andtwo hours; commonly fifteen to 30 minutes are suflicient to reduceCompounds IVa or IV!) to the corresponding 21-hydroxyor21-acyloxy-3u-hydroxyl4- pregnen-20-one (Va and Vb). Products V areisolated from the reaction mixture by neutralizing the reaction mixturewith a dilute acid, for example, dilute hydrochloric acid and extractingCompound V with a waterimmiscible solvent such as methylene dichloride,chloroform, carbon tetrachloride, ethylene dichloride, Skellysolve Bhexanes, and the like. The extracts are then treated in conventionalmanner, that is products (Vb), 21-acyloxy-3a-hydroxy-14-pregnen-20-onesare obtained by removing through distillation the solvent and purifyingthe residue in conventional manner such as by recrystallization andchromatography.

The thus-obtained 21-acyloxy-3a-hydroxy14-pregnen 20-one (Vb) is thenesterified in the 3-position with an arylsulfonic acid. For this purpose2.1-acyloxy-3u-hydroxy-14-pregnen-20-one is dissolved in pyridine or inan inert solvent such as benzene, toluene, dioxane, acetone, and thelike, and thereto is added an arylsulfonic acid halide. Arylsulfonylhalides useful for this purpose include particularly the benzenesulfonylchlorides such as para-toluenesulfonyl chloride,para-chlorobenzenesulfonyl chloride, para-bromobenzenesulfonyl chloride,para-nitrobenzenesulfonyl chloride and B-naphthylsulfonyl chloride andfi-naphthylsulfonyl bromide with para-toluenesulfonyl chloridepreferred. The esterification with the selected arylsulfonyl halide ispreferably carried out at room temperature by maintaining the mixturefor a period of three to 24 hours whereafter the ester is isolated byconventional methods, such as precipitation from the mixture after waterhad been added, filtration and recrystallization to purify the materialand to obtain pure 21-acyloxy-3a-arylsulfonyloxy-14-pregnen- 20-one(VI).

The 21-acyloxy-3u-(arylsulfonyloxy)- 14 pregnen-ZO- one (VI) isconverted to the corresponding 3B-isomer esters by treating it insolution of a carboxylic acid containing from one to eight carbon atoms,inclusive, and having a melting point below fifty degrees centigradewith the corresponding sodium or potassium hydrocarbon carboxylic acidacylate. In the preferred embodiment of the invention the reactionmixture is boiled for a period of one to four hours and thereafter theexcess carboxylic acid is removed. Evaporation aided by an air stream orvacuum distillation are the preferred means to remove the excess organicacid. The residue is purified by conventional means such asrecrystallization from suitable solvents and/ or chromatography whenindicated. In this manner a 3,8-ester, the 3,8,21-diacyloxy-l4-pregnen-ZO-one (VIIb) is obtained. Saponification of the 3 3,21-diacyloxy-14-pregnen-20-one with sodium carbonate or dilute sodiumhydroxide in aqueous alcoholic solution under nitrogen produces thecorresponding 3,8,21-diol, 313,2 l-dihydroxy-14-pregnen-20-one (VIIa)The thus obtained 3,8,21-diacyloxy-14-pregnen-20-one (VIIb), dissolvedin a suitable organic solvent such as methylene dischloride, ethylenedichloride, dioxane, tertiary butyl alcohol, chloroform, carbontetrachloride and mixtures thereof is admixed with hypohalus acid orpreferably with a reagent releasing a hypohalus acid such as anN-halohydrocarbon acid amide, for example, N-bromoacetamide,N-chloroacetamide, N-bromosuccinimide, N- chlo-rosuccinimide, whendissolved in an aqueous mineral acid acidified solution. In thepreferred embodiment, two to three mole equivalents of theN-halohydrocarbon carboxylic acid amides are used for each molarequivalent of 35,2l-diacetoxy-14-pregnen-20-one (VIIb To acidify thesolution of the N-halohydrocarbon carboxylic acid amide, one to fiveacid equivalents of a mineral acid such as sulfuric, perchloric orperiodic acid are used for each molar equivalent of theN-halohydrocarbon carboxylic acid amide. After a reaction period of fromfive to sixty minutes any further reaction is prevented, for example, byadding sodium sulfite, dissolved in water. The solution is thenconcentrated in vacuo and poured into a large amount of chipped ice. Theprecipitate thus obtained is removed by filtration and if so desiredpurified by recrystallization to give pure3,3,21-diacyloxy-15fi-hydroxy-l4u-bromopregnan-20-one.

The thus-obtained 3,8,21-diacyloxy-15;3-hydroxy-14abromopregnan-20-one(VIIIb), carefully dried with about an equal amount by weight ofiodinated zinc granules in vacuo at about degrees centigrade for aperiod of eighteen hours, was dissolved in an inert organic solvent suchas dioxane, Skellysolve B hexanes, methylene dichloride, ethylenedichloride 7 chloroform, carbon tetrachloride, and thereafter admixedwith ethyl bromoacetate. in the preferred procedure the ethylbromoacetate is added dropwise under stirring with regulation of thespeed of reaction by heating or cooling the reaction flask as deemednecessary, usually cooling at the onset of the reaction and heatingtoward the end of the reaction. The thus-obtained 3,8-acyloxy 15Bhydroxy-l4wbromo-14a-card-20 (22)-enolide (IX) is isolated from thereaction mixture by conventional procedures such as extraction withwaterimmiscible solvents, for example, ether, methylene dichloride,ethylenedichloride, chloroform, carbon tetrachloride, and the like, andrecrystallization from suitable organic solvents such as ethyl acetate,acetone, Skellysolve B hexanes, methanol and mixtures of such solvents,and the like.

The thus-obtained bromohydrine compound,3fi-acyloxy-lB-hydroxy-14a-bromo-14m-card 20(22) enolide is converted tothe corresponding 14,15-oxido compound (Xb) by reacting3fl-acetoxy-15,8-hydroxy-14a-bromol4cecard-20(22)-enolide with a basesuch as potassium acetate, sodium acetate, soda lime, or basichydroxides such as hydroxides of sodium, potassium, calcium, barium, andthe like. The reaction is usually carried out in an organic solvent. Ifthe selected condensation reagent is strongly basic such as sodium orpotassium hydroxide the solvent must be of such a nature as not toundergo basic condensation, for example, dioxane, tertiary butylalcohol, and the like. If weaker bases, potassium acetate or sodiumacetate, are selected, then acetone, ethyl alcohol, ethyl acetate, oreven chlorinated hydrocarbons may be used. Preferably the reaction iscarried out at the reflux temperature of the solvent. At the end of thereaction period the reaction mixture is suitably poured into excess ofwater, the resulting precipitate collected on fi ter, and the materialpurified by recrystallization from solvents, for example, acetone,methanol or Skellysolve B hexanes to give pureBfi-acyloxy-l4,l5-oxido-20(22)-cardenolide (Xb).

The thus-obtained 3;3acyloxy-14( 15 -oxido-20(22) cardenolide (Xb) ishydrolyzed to the corresponding 3- alcohol (Xa) by using a fungusselected from the genus Rhizopus, suitably Rlzz'zopus slzanghniensis(ATCC 10329) in an anaerobic short term fermentation, with medium andconditions of agitation as described in US. Patent 2,602,769 and morespecific-ally in the example of the instant application. After thefermentation the thus-produced 36 hydroxy 14(15)oxido-20(22)-cardenolide (Xa) is separated and isolated from thefermentation medium by conventional means, such as extraction,recrystallization, chromotography or combinations thereof as describedin detail in the example.

The thus-obtained 3Bhydroxy'-14( 15 -oxido-20(22)- cardenolide (Xa) istaereafter reduced with a metallic hydride, such as sodium or potassiumborohydride. The reduction is generally carried out at a temperature between zero and 30 degrees centigrade in dioxane, tetrahydrofuran orother solvent solutions. The lower temperature range between zero andten degrees is preferred and in this case the time of reaction will bebetween two and ten hours with a period from four to six hourspreferred. Thereafter the reaction product is isolated in conventionalmanner usually by first acidifying the solution with dilute acid,concentrating the thus obtained acidic reaction mixture to a smallvolume, diluting with excess water and extracting with a waterimmiscible solvent such as chloroform, Skellysolve B hexanes, carbontetrachloride, methylene dichloride, and the like. After the solvent isremoved from the extract a solid residue is obtained which consisted oftwo major components digitoxigenin [3B,l45-dihydroxy-20(22)-cardenolide]and its '15/8-hydroxy isomer which were separated by chromawa nhy- Thefollowing examples are illustrative of the process of the presentinvention and are not to be construed as limiting.

PREPARATION 1 1 5 a-hydroxy progesterone monium sulfate, 0.2 gram ofzinc sulfate, 0.05 gram of ferrous sufate, and one gram of sodiumacetate diluted to one liter with industrial tap water. Twelve liters ofthis heat-sterilized medium was inoculated with spores of Penicilliumurticae, ATCC 10120. Into this, there was dispersed a solution of threegrams of progesterone in milliliters of acetone. Fermentation proceededat room temperature for 48 hours with agitation and areation at a rateof one liter per minute. The whole beer containing mycelium wasextracted four times, each time with three liters of methylene chloride.The combined methylene chloride extract was washed twice, each time withone-tenth by volume portions of a two percent aqueous solution of sodiumbicarbonate and then twice with one-tenth by volume portions of water.The methylene chloride extract was dried with anhydrous sodium sulfateand then freed of solvent on a steam bath. The residue was dissolved in300 milliliters of benzene and chromatographed over 150 grams of alumina(hydrochloric acid washed, water washed, and dried at 1.20 degreescentigrade for four hours) using 300-milliliter portions of developingsolvent as indicated in Table I.

Fractions 19 and 20 were combined and dissolved in methylene dichloride,mixed with a small proportion of Magnesol magnesium silicate andfiltered. The filtrate was concentrated to five milliliters and thenmixed with fifty milliliters of ether to give 746 milligrams of acrystalline precipitate. Recrystallization from 25 milliliters of ethylacetate and then from five milliliters of methylene chloride by theaddition of fifty milliliters of ether produced 572 milligrams ofcrystals melting at 223 to 230 degrees centigrade. Two furtherrecrystallizations from methylene dichloride ether yielded 302milligrams of l5ot-hydroxyprogesterone having a melting point of 230 to234 degrees centigrade, [ch -224 degrees (C. 1.0 in chloroform),

xx, 242; E: 15,400

Analysis.-Calculated for C I-1 0 C, 76.32; H, 9.15. Found: C, 76.79; H,9.49.

The infrared spectrum confirmed the structure.

TABLE I Fraction Solvent Eluate Solids,

Milligrams 1 benzene 98 2.. do 35 3.- benzene-ether 20:1 24 4.- .do 54benzene-ether 10:1 123.5 ..do.. 177 206 245 191 142 68 o 64etherehlorof0rm 10:1. 13 o.. 12 ether-chloroform 1:1 17 ..do... 71 do104 18. do 93 19'. hlorofoi 42 20. do..-" 632 21. ...do 301 22. ..d0 v34 23... chl0rot3rrnacetone 20:1 12 24... acetone 51 25-27,methan01...,,. 181

9 PREPARATION 2 JS-aeetoxyprogesterone A mixture of 100 milligrams of15-hydroxyprogesterone, one milliliter of acetic anhydride and onemilliliter of pyridine was maintained at room temperature for 24 hours.It was then quenched with 100 milliliters of ice water and extractedwith chloroform. The chloroform extracts were washed twice with fivepercent hydrochloric acid, twice with five percent sodium carbonate andthree times with water. Evaporation of the washed extract gave 105milligrams of oily residue which was crystallized from ethyl acetate andrecrystallized from methanol to yield 80.5 milligrams ofIS-acetoxyprogesterone having a melting point of 181 to 183 degreescentigrade. Structure was confirmed by infrared spectrum; A maximum inalcohol of 241 millimicrons, E of 16,800; and an optical rotation ofplus 178 degrees at a con- 'centration of 1.0 in chloroform.

PREPARATION 3 lu-formyloxyprogesterone In the same manner as Preparation2, using an excess of formic acid in place of acetic anhydride produced1S-formyloxyprogesterone.

PREPARATION 4 oz-benzoxyprogesterone In the same manner as inPreparation 2, using benzoyl chloride in place of acetic anhydride,produced 15a-benzoyloxyprogesterone.

In a similar manner other esters of 15a-hydroxyprogesterone are preparedaccording to the acylation procedure illustrated above or by reactionswith ketene, ketenes of selected acids, selected acids, acid anhydridesor acid halides such as acid chlorides and acid bromides in an organicsolvent such as pyridine, or the like. Representative of suchISa-acyloxyprogesterones are the acylates of IS-hydroxyprogesterone ofhydrocarbon car boxylic acid esters of saturated or unsaturatedaliphatic, carboxylic or cycloaliphatic, arylalkyl, alkaryl, monoordipolycarboxylic acids having between one to eight carbon atoms,inclusive, such as, for example, the propionyloxy, butyryloxy,isobutyryloxy, valeryloxy, isovaleryloxy, hexanoyloxy, heptanoyloxy,octanoyloxy, benzoyloxy, phenylactetoxy, toluyloxy,B-cyclopentylpropionyloxy, acrylyloxy, hemisuccinyloxy, hemimaleyloxy,hemitartaryloxy, dihydrogencitryloxy, dimethyland trimethylacetoxy, orthe like. The acids may also contain non-interfering substituents suchas mono, or poly halo, chloro, bromo, hydroxy, methoxy, and the likegroups if so desired.

EXAMPLE 1 4,14-pregnadiene-3,ZO-diorze from 15a-acetoxypr0- gesteroneOne gram of l5a-acetoxyprogesterone was placed in a tube ten centimeterslong and pyrolyzed in a combustion furnace heated to 300 degreescentigrade. The compound was distilled in a nitrogen stream through thetube. The oily distillate was thereupon taken up in chloroform, washedtwice with five percent sodium carbonate solution and three times withwater. The chloroform solution was then dried with anhydrous sodiumsulfate and the solvent removed. Thereupon 854 milligrams of an oilyresidue was obtained. This residue was dissolved in 200 milliliters ofSkellysolve B hexane hydrocarbons and chromatographed over 40 grams ofalumina. Fractions of 200 milliliters were collected as shown in TableII.

Eluate fractions 7 through 10 were combined and recrystallized threetimes from ethanol to give 350 milligrams of 4,14-pregnadiene-3,20-dioneof melting point 144 to 147 degrees centigrade and rotation [041 +128 75degrees.

10 Analysis.-Calculated for 0 11 0,: C, 80.73; H, 9.03. Found: C, 80.58;H, 8.99.

TABLE II Fraction Solvent Eluate Solids,

' Milligrams 1 Skellysolve B (petroleum ethe 2 do 3 -do 3.0 4Skellysolve Bbenzene 1: 14. 0 5.. -..-do 6.0 6. ..-do.-- 15.0 7. Benzene117. 5 8.. --..-d0 123.0 9 d0-.. 85.5 10 Benzene10% ether. 214.0 11..-d0 52.5 ..do 6.0 Benzene-30% ether EXAMPLE 2 4,14-pregnadiene-3,20dione from 15u-pr0pi0nyl0xyprogesterone In the same manner as given inExample 1, ISu-propionyloxyprogesterone instead of theISa-acetoxyprogesterone was pyrolyzed at a temperature of 350 degreescentigrade to yield 4,14-pregnadiene-3,20-dione.

EXAMPLE 3 3,20-dione.

EXAMPLE 4 4,14-pregnadiene-3,20-di0ne from 14a-liydr0xypr0- gesteroneThree grams of l4vr-hydroxyprogesterone was dissolved in 50 millilitersof acetic anhydride. To this solution there was added 4.5 grams offreshly fused potassium acid sulfate. The mixture was heated on thesteam bath for thirty minutes, then cooled with ice. The cooled solutionwas diluted with 300 milliliters of ice water and maintained overnight.The aqueous solution was then extracted with chloroform four times.Chloroform extracts were washed three times with a five percent sodiumcarbonate solution and four times with water. Upon concentration of thechloroform extracts, 2.7 grams of a waxy residue was obtained.

The residue was dissolved in 150 milliliters of Skellysolve B hexanehydrocarbon and purified by chromatog- TABLE III Fraction Solvent EluateSolids,

Milligrams Skellysolve B Skellysolve B% benzene Benzene Benzene-30%ether. Benzene50% ether.

I Methanol....

spasms Fractions 13 through 16 were combined, atotal of 532 milligrams,recrystallized from ethyl acetate, sublimed at 0.05 millimeter pressureand at 135 degrees centigrade, and recrystallized once more frommethanol to give 291.5 milligrams of 4,14-pregnadiene-3,20-dione ofmelting point 144 to 146 degrees centigrade.

EXAMPLE 21 -hydr0a cy4,14-pregnodiene-3,20-dione Eight liters of amedium were prepared containing per liter 30 grams of malt extract, 50grams of Cerelose dextrose, two grams of dihydrogen potassium phosphate,0.5 gram of magnesium sulfate heptahydrate, and 0.03 gram of zincsulfate heptahydrate. Eight liters of this heat sterilized medium wereinoculated with Ophiobolus herp otrichus and grown for a period of 24hours at a rate of aeration equal to one liter per minute and a rate ofagitation of 200 r.p.m. To this fermentation mixture containing a24-hour growth of Ophiobolus herpotrichus, two grams of4,14-pregnadiene-3,20 dione dissolved in acetone was added. .After anincubation period of 48 hours at a temperature between 26 and 30degrees, the reaction mixture was extracted with methylene dichloride togive 3.55 grams of an oily extractive which crystallized upontrituration with ether. The steroid compound was recovered after athree-fold trituration of the extractives and decantation of the ethermother liquors. Thus, 1.2 grams of 2l-hydroxy-4,14-regnadiene-lZO-diO-ne of melting point 182 to 184 degrees centigrade wasobtained.

EXAMPLE 6 21 -acet0xy-4,I4-pregnadiene-3,20-dione One hundred milligramsof 21-hydroxv-4,14-pregnadiene-3,20-dione was dissolved in twomilliliters of absolute pyridine and one milliliter of acetic anhydride.The mixture was maintained for a period of two hours, then diluted withWater, and chilled, and the resulting precipitate was collected on afilter. The crystals were redissolved in two milliliters of acetone andprecipitated from acetone by the addition of hexane to the acetonesolution. The thus-obtained recrystallized 21-acetoxy-4,14-pregnadiene-3,20-dione had a melting point of 142 degreescentigrade.

EXAMPLE 7 .21 -propionyIoxy-4,1 4-pregnadiene-3,20-dione In the samemanner as given in Example 6, using the equivalent proportion ofpropionic anhydride in place of acetic anhydride produced21-propionyloxy-4,14-pregnadiene-3,20-dione.

EXAMPLE 8 21 -benzoyl0xy-4,14-pregnadiene-3,20-dione In the same manneras given in Example 6, using the equivalent proportion of benzoylchloride in place of acetic anhydride produced21-benzoyloxy-4,14-pregnadiene-3,20-dione.

In a similar manner as .given in Examples 6 thru 8, other esters of21-hydroxy-4,14-pregnadiene-3,20-dione are prepared by reacting21-hydroxy-4,14-pregnadiene- 3,20-dione with the selected acidanhydride, ketene, ketene of a selected acid or with a selected acidhalide in pyridine or with other acylating agents as previouslydescribed in this specification. Representative esters of 21-hydroxy-4,14-pregnadiene-3,20 dione include the 2l-acyloxy: formyloxy-,butyryloxy-, isobutyryloxy-, valeryloxy-,, isovaleryloxy-, hexanoyloxy-,heptanoyloxy-, octanoyloxy-, benzoyloxy-, (fJ-cyclopentylpropionyloxy)-,dimethylacetoxy-, trimethylacetoxy-, phenylacetoxy-, toluyloxy-,anisoyloxy-, gallyloxy-, salicyloyloxy-, cinnamyloxy-, hernisuccinyloxy,hemitartaryloxy-., dihydrogencitryloXy-, maleyloxy-, 'fumaryloxy-,crotonyloxy-, acrylyloxy-, (flmethylcrotonyloxy)-,cycloheXane-carbonyloxy-, chloroacetoxy-, dichloroacetoxy-,trichloroacetoxy-, bromoacet- 21 -hydroxy-14-pregnarm-3,20-dione Apalladium zinc carbonate zinc oxide catalyst was prepared as follows:eleven grams of anhydrous zinc chloride was dissolved in milliliters ofwater at seventy degrees centigrade, and a twenty percent aqueoussolution of sodium carbonate was added in excess, with constantstirring, to form a precipitate of zinc carbonate. The precipitate wasfiltered, washed free of alkali with Warm water, and resuspended in 100milliliters of water to form a slurry. To this aqueous slurry was addedfive milliliters of a palladium chloride solution containing 0.5 gram ofpalladium, and then one milliliter of 37 percent aqueous formaldehydesolution. The resulting red-brown colored mixture was warmed on a steambath to about sixty degrees centigrade and a thirty percent aqueoussolution of sodium hydroxide then added dropwise, with continualstirring, until the pH of the mixture reached nine, at which pointpalladium precipitated. The blackcolored precipitate was washed bydecantation with ten successive fifty-milliliter portions of water andthen filtered under suction. The precipitate on the filter funnel waswashed six times, dried by suction, and heated in an oven at 210 degreescentigrade for a period of eleven hours. Six and eight-tenths grams of abrown colored catalyst consisting of palladium supported on a zinccarbonate-zinc oxide mixture was obtained.

1.5 grams of this catalyst was introduced into ten millilters ofmethanol and the mixture hydrogenated for a period of one hour. To thissolution was then added 1.5 grams of21-hydroxy-4,14-pregnadiene-3,20-dione, dissolved in 100 milliliters ofmethanol. This reaction mixture took up 1.1 moles of hydrogen within 15minutes. The reaction was interrupted and the solution was filtered andthe filtrate put through a column of activated carbon and diatornaceousearth 1:1 (60 grams). The desired product was recovered by elution withnine fractions of BOO-milliliters of acetone to give 1.25 grams ofcrystals of melting point to 157.

EXAMPLE l0 21-acetoxy-14-pregnene-3,20-dione To a solution of 80milligrams of 21-hydroxy-l4-pregmeme-3,20 dione in 1.5 milliliters ofpyridine was added one milliliter of acetic anhydride and the mixtureallowed to stand for a period of three hours. Thereafter the reactionmixture was diluted With fifty milliliters of ice Water and theresulting aqueous solution filtered, obtaining in this manner crystalsof 21-acetoxy-14-pregnene- 3,20-dione which were recrystallized frommethanol to give pure 21-acetoxy-14-pregnene-3,20dione of melting point154 to 15,7.

EXAMPLE 11 In the same manner as given in Example 10, using theequivalent proportion of butyric anhydridein place of acetic anhydrideproduced 2l-butyryloxy-14-pregnene- 3,20-dione.

EXAMPLE 13 21-hexanoyloxy-J4-pregnene-3,20-dione In the same manner asgiven in Example 10, using the equivalent proportion of hexanoic acidanhydride in place of acetic anhydride produced21-hexanoyloxy-l4-pregnene3,20-dione.

EXAMPLE 14 21-benz0yl0xy-14-pregnene-3,20-di0ne In the same manner asgiven in Example 9, hydrogenating21-benzoyloxy-4,14-pregnadiene-3,20-dione instead of21-hydroxy-l4-pregnene-3,20 dione with hydrogen in the presence of apalladium-zinz oxid'e catalyst prepared as described before yields2l-benzoyloxy-14-pregnene- 3,20-dione.

EXAMPLE 15 21-pr0pi0nyl0xy-14-pregnene-3,20-dione In the same manner asgiven in Example 9, hydrogenating21-propionyloxy-4,l4-pregnadiene-3,20-dione instead of2l-hydroxy-14-pregnene-3,20-dione with hydrogen in the presence of apalladium-zinc oxide-zinc carbonate catalyst yields2l-propionyloxy-14-pregnene-3,20- dione.

EXAMPLE 16 21-t0luyloxy-14-pregnene-3,20-dione In the same manner asgiven in Example 9, hydrogenating21-toluyloxy-4,l4-pregnadiene-3,20-dione instead of2l-hydroxy-l4-pregnene-3,20-dione with hydrogen in the presence of apalladium-zinc oxide-zinc carbonate catalyst prepared as describedbefore yields 21-toluyloxy-l4- pregnene-3 ,20-dione.

EXAMPLE 17 21-anisoyloxy 14-pregnene-3,20-di0ne In the same manner asgiven in Example 9, hydrogenating2l-anisoyloxy-4,14-pregnadiene-3,20-dione instead of2l-hydroxy-14-pregnene-3,20-dione with hydrogen in the presence of apalladium-zinc oxide-zinc carbonate catalyst yields2l-anisoyloxy-14-pregnene-3,20- dione.

In a manner similar to Examples 10 through 13, by reacting21-hydroxy-l4-pregnene-3,20-dione with the selected anhydride, ketene,ketene of a selected acid or selected acid halide in pyridine or withother acylating agents and solvents as previously described, or byhydrogenation of the corresponding2l-acyloxy-4,14-pregnadiene-3,20-dione (Examples 6-8) as described inExamples 14 thru 17, the corresponding 2l-acyloxy-14-pregnene-3,20-dioues are obtained.

EXAMPLE 18 21 -acet0xy-3u-hydr0xy-1 4-pregnen-20-0ne A solution of 1.2grams of 21-acetoxy-14-pregnene- 3,20-dione, dissolved in 7.5milliliters of dioxane, redistilled over lithium aluminum hydride, wascooled to a temperature of ten degrees Centigrade. To this solution wasadded, under vigorous stirring, 38.9 milligrams of sodium borohydride,dissolved in 0.6 milliliter of 0.1 normal sodium hydroxide solution. Atthe same time 6.0 milliliters of cold, deionized water was added. Aftera reaction time of fifteen minutes at a temperature of twenty degreescentigrade, the reaction mixture was neutralized with dilutehydrochloric acid and extracted with methylene dichloride. The methylenedichloride extracts were purified by chromatography over Florisilanhydrous magnesium silicate and recr stallized to give 677 milligramsof 21-acetoxy-3a-hydroxy-l4-pregnen-20-one of melting point 198 degreescentigrade.

EXAMPLE 19 21 -benz0yloxy-3a-hydr0xy-14-pregnen-20-one In the samemanner as given in Example 18, reducing21-benzoyloxy-14-pregnene-3,20-dione with potassium borohydride yielded2l-benzoyloxy-3a-hydroxy-14- pregnen-ZO-one.

EXAMPLE 20 21-pr0pi0nyI0xy-3a-hydrOxy-M-pregnen-ZO-one In the samemanner as given in Example 18, reducing21-propionyloxy-l4-pregnene-3,20-clione with potassium borohydrideyielded 21-propionyloxy-3tx-hydroxy-14-pregnen-20-one.

EXAMPLE 21 21 (B-cyclopentylpropionyloxy) -3a-hydr0xy-14- pregnen-Z0-one In the same manner as given in Example 18, reducing2l-propionyloxy-14-pregnene-3,20-dione with sodium borohydride yielded21-propionyloxy-3a-hydroxy-14-pregnen-20-one.

EXAMPLE 22 21-t0luyloxy-3a-hydroxy-14-pregnen-20-one In the same manneras given in Example 18, reducing 2l-toluyloxy-14-pregnene-3,20-dionewith sodium borohydride yielded 21-toluyloxy-3a-hydroxy-14-pregnen-20-one.

In a manner similar to Examples 18 through 22, using the startingmaterials of Examples 10 through 17 with sodium or potassium borohydrideis productive of the corresponding 21 acyloxy 30c hydroxy 14pregnen-ZO-ones.

EXAMPLE 23 3a,21-diacet0xy-14-pregnen-20-one To a solution of 50milligrams of 21-acetoxy-3a-hydroxy-14-pregnen-20-one in one milliliterof pyridine was added 0.5 milliliter of acetic anhydride. The solutionwas maintained for a period of six hours, poured into twenty millilitersof ice water, chilled in a refrigerator at plus five degrees centigradefor a period of fifteen hours, thereafter filtered and the obtainedcrystals recrystallized from a mixture of acetone and methanol. The thusobtained 30,2l-diacetoxy-l4-pregnen-2O-one had a melting point of 205 to207 degrees centigrade.

In a manner similar to Example 23 by esterifying 21-.acyloxy-3a-hydroxy-14-pregnen-20'ones with selected acid anhydrides oracid halides, other representative 30,21- diacyloxy-l4-pregnen-20-onesare obtained wherein the acyloxy group of the 30c and the 21-positionmay be the same or dissimilar. Illustratively, other 3a,21-diacyloxy-14-pregnen-20-oues include: 3a,21-dipropionyloxy-, 3oz,21-dibutyryloxy-, 3a,2l-divaleryloxy-, 3u,21-dihexanoyloxy-,3a,2l-dibenzoyloxy-, 3a,2l-diphenylacetoxy-, 3aacetoxy 21 benzoyloXy-,Bot-acetoxy 21-cinnamyloxy-, 3a acetoxy 21 salicyloyloxy-, 3ozpropionyloxy 2l-acetoxy-, 3wbutyryloxy-Z1-benzoyloxy-,3a-hexanoyloxy-21-phenylacetoxy-14-pregnen-20-one, and the like.

EXAMPLE 24 3u,21-dihydr0xy-14-pregnen-20-one Toa solution of fiftymilligrams of 2l-acetoxy-3ahydroxy-l4-pregnen-20-one in two millilitersof ethyl al cohol was added fifty milligrams of sodium bicarbonate. Themixture was refluxed in a nitrogen atmosphere for a period of 48 hours,filtered through filter paper, and poured into twenty milliliters of icewater. The oily residue was recrystallized from acetone and SkellysolveB hexane hydrocarbons to give 30,2l dihydroxy-l4-pregnen- 20-one.

In a manner similar to Example 24 other 2l-acyloxy-3a-hydroxy-l4-pregnen-20-ones and 3rx,2l-diacyloxy-14- pregnen-ZO-oneslike those shown in Example 19-23 are hydrolyzed to give311,2l-dihydroxyl4-pregnen-20-one.

1s EXAMPLE 25 21-acetoxy-3a- (para-toluenesulfonyloxy -14-pregnen-20-one To a solution of 2.1 grams of 21-acetoxy-3a-hydroxy-14-pregnen-20-one in 25 milliliters of dry pyridine was added 1.5 gramsof para-toluenesulfonyl chloride. After maintaining at room temperaturefor a period of fifteen hours the solution was diluted with 300milliliters of water. The precipitate thus obtained was separated byfiltration, washed with water several times, and recrystallized threetimes with ethyl acetate-chloroform 1:1 to give 1.95 grams of21-acetoxy-3u-(para-toluenesulfonyloxy)-14-pregnen-20-one of meltingpoint 212 to 215 degrees centigrade.

EXAMPLE 26 21 -benzyl0xy-3a- (para-toluenesulfonyloxy -14-pregnen-20-0ne In the manner given in Example 25, 2lbenzoyloxy-3a-hydroxy-14-pregnen-20-one was reacted with paratoluenesulfonylchloride to produce 2l 'benzoyloxy-3u-(para-toluenesulfonyloxy)-14-pregnen-20-one.

EXAMPLE 27 21-benzoyloxy-3a-benzenesulfonyloxy-J4-pregnen-20-one In thesame manner as given in Example 25, 21- benzoyloxy-3ot-hydroxy-14-pregnen-20-one is reacted with benzenesulfonyl chloride toproduce 21-benzoyloxy-3abenzenesulfonyloxy-14-pregnen-20-one.

EXAMPLE 28 21-pr0pi0nyl0xy-3a-(para-chlorobenzenesulfonyloxy)-14-pregnen-ZO-one In the same manner as given in Example 25, 21-propionyloxy-3u-hydroxy-14-pregnen-20-one was treated withpara-chlorobenzenesulfonyl bromide to give 21- propionyloxy 3a (parachlorobenzenesulfonyloxy)- 14-pregnen-20-one.

EXAMPLE 29 21-toluyloxy-3ot-(para-bromobenzenesulfonyloxy)-14-pregnen-ZO-one In the same manner as given in Example 25, 21-toluyloxy-3ot-hydroxy-14-pregnen-20-one was treated withpara-bromobenzenesulfonyl bromide to give 21-toluyloxy- 3 ot-(parahromobenzenesulfonyloxy) -14-pregnen-20-one.

EXAMPLE 30 21-butyryl0xy-3a- (/EZ-naphthylsulfonyloxy -14-pregnen-20-0ne In the same manner as given in Example 25, 21-butyryloxy-3a-hydroxy-14-pregnen-20-one was treated withB-naphthylsulfonyl bromide to give 2l butyryloxy-3afi-naphthylsulfonyloxy) -14-pregnen-20'one.

EXAMPLE 31 21-propi0nyl0xy-3a-(para-t0luenesulfonyloxy)-14-pregnen-ZO-one 1'6 EXAMPLE 32 35,21-diacetoxy-14-pregnen-20-one Asolution of 1.55 grams of21-acetoxy-3a-(para-toluen'esulfonyloxy)-14-pregnen-20-one, 1.5 grams ofanhydrous sodium acetate and fifty milliliters of glacial acetic acidwere refluxed for a period of two hours at atmospheric pressure.Thereafter the solution was concen-' trated in an air stream, and theresidue after dispersion in water, extracted with chloroform. Thechloroform extracts were Washed three times with five percent sodiumhydroxide and three times with water. The residual extractives, 1.60grams, were purified by chromatography over grams of hydrochloric acidwashed alumina. Ether-five percent chloroform eluted 755 milligrams of21-acetoxy-3,14-pregnadien-20-one and ether-fifty percent chloroform andchloroform eluted 805 milligrams of 36,21-diacet0xy-14-pregnen-20-onewhich was recrystallized twice from ethanol to give a melting point of178 to 180 degrees centigrade.

EXAMPLE 33 318 acetoxy 21 propionyloxy 14 pregnen 20 one In the samemanner as given in Example 32, 21- propionyloxy3a-(para-toluenesulfonyloxy)-14-pregnen- 20-one was refluxed in glacialacetic acid in the presence of anhydrous potassium acetate to give3fl-acetoxy-21- propionyloxy-l4-pregnen-20-one.

EXAMPLE 34 3fi-butyryloxy-ZI-propionyl0xy-14-pregnen-20-0ne In the samemanner as given in Example 32, 21- propionyloxy 3a(para-chlorobenzenesulfonyloxy)-14- pregnen-20-one was treated withbutyric acid and potas sium butyrate to give3fi-b-utyryloxy-21-propionyloxy-l4- pregnen-ZO-one.

EXAMPLE 35 3,8- 1r0p ionyloxy-ZI -benzoylox'y-14-pregnen-20-0ne In thesame manner as given in Example 32,21-benzoyloxy-3a-benzenesulfonyloxy-14-pregnen-20-one is refluxed inpropionic anhydride in the presence of sodium propionate to give3fl-propionyloxy-21-benzoyloxy-14- pregnen-ZO-one.

EXAMPLE 36 .ifl-valeryloxy-Zl -toluyl0xy-14-pregnen-20-0ne In the samemanner as given in Example 32,21-toluyloxy-3a-(para-bromobenzenesulfonyloxy) 14 pregnen- 20-one isrefluxed in valeric anhydride in the presence of potassium valerate togive 3 3-va1eryloxy-21-toluyloxy-14- pregnen-20-one.

EXAMPLE 37 3fi-acetoxy-21-butyryIoxy-M-pregnemZO-One oxy l4-pregnen20-one, Sfi-actoxy 21-heptanoyloxy- 14-pregnen 20 one,3B-acetoxy-21-octanoyloxy 14- pregnen 2O one,3fl-acetox-y-21-(B-cyclopentylpropionyloxy)-14-pregnen 20 one, 36propionyl0xy-21-cinnamyloxy 14 pregnen-ZO-one, 3 3-butyryloxy-2l-anisoyloxy-14 pregnen 20 one, 3,8-valeryl0xy-2l-gallyloxy- 14 pregnen20 one, 3 8-hexanoyloxy-2l-hernisuccinylp y-l4-pregnen-20-one, andthelike.

EXAMPLE 38 3,9,21-dihydrxy-14-pregnen-20-one To a solution of 500miligrams of 35,21-diacetoxy-14- pregnen-ZO-one in seven milliliters ofmethanol and three milliliters of water was added 500 milligrams ofsodium carbonate. The reaction mixture was allowed to stand at roomtemperature, twenty to thirty degrees centigrade, for a period of 24hours. Thereafter the mixture was diluted in fifty milliliters of waterand the thus obtained precipitate was recrystallized from ethyl acetateto give crystals of 3fi,2l-dihydroxy-14-pregnen-20-one.

EXAMPLE 39 35,21 diacetoxy-ISB-hya'roxy-J4u-br0mopregnan-200ne Asolution of 2.5 grams of 3,8,2l-diacetoXy-14-pregnen- 20-one in 100milliliters of methylene dichloride and 200 milliliters of tertiarybutylalcohol was prepared by vigorous stirring at room temperature. To thissolution was added 6.5 milliliters of seventy percent perchloric acid,dissolved in fifty milliliters of water, and 1.1 grams ofN-bromoacetamide, dissolved in 12 milliliters of tertiary butyl alcohol.The mixture was stirred for a period of ten minutes at a temperature oftwenty-five degrees centigrade. Thereafter 1.2 grams of sodium sulfite,dissolved in sixty milliliters of water, was added. The solution wasthen concentrated to one-third of the origi nal volume in vacuo andpoured thereafter into 300 milliliters of ice with stirring. Theprecipitate which separated was collected by filtration and dried invacuo to give 2.45 grams of crude3,9,2l-diacetoxy-lSfl-hydroxyl4a-bromopregnane-20-one which was twicerecrystallized from methyl alcohol.

EXAMPLE 40 3,8 acetoxy 21 propionyloxy 15,8 hydroxy14abromopregnan-ZO-one In the manner given in Example 39, SB-acetoxy-ZLpropionyloxy-l4-pregnen-20-one, dissolved in methylene dichloride andtertiary butyl alcohol, was treated with an acidified solution ofN-bromosuccinimide to yield 3,8- acetoxy 21 propionyloxy 15,3 hydroxy14a bromopregnen-ZO-one.

EXAMPLE 41 3,8 propionyloxy 21 benzoyloxy 15,8 hydroxy14abromopregnan-ZO-one In the manner given in Example 39,3,6-propionyloxy- 2l-benzoyloxy-l4-pregnen-20-one, dissolved inchloroform and tertiary butyl alcohol, was treated with N-bromoacetamideacidified with dilute sulfuric acid to yield 3,6- propionyloxy 21benzoyloxy 15,8 hydroxy 14abromopregnen-ZO-one.

EXAMPLE 42 3e valeryloxy 21 toluyloxy 15B hydroxy 14abromopregnan-ZO-oneIn the manner given in Example 39, 33-valeryloxy-2ltoluyloxy-l4-pregnen-20-one, dissolved in methylenedichloride and tertiary butyl alcohol was treated with N-bromoacetarnideacidified with perchloric acid to yield 33 valeryloxy 21 toluyloxy 15 8hydroxy 14mbromopregnan-ZO-one.

EXAMPLE 43 3fi,21-diacetoxy-1Sp-hydroxy-I4u-chloropregnan-20-one In themanner given in Example 39, 35,2l-diacetoxy- 14-pregnen-20-one,dissolved in tertiary butyl alcohol, was treated withN-chlorosuccinimide acidified with dilute sulfuric acid to yield35,2l-diacetoxy-lSfi-hydroxy- 14u-chloropregnan-20-one.

In the same manner as shown in Examples 39 through 43, othercorresponding 35,2l-diacyloxy-lSfi-hydroxy- 14a-halopregnen-20-ones areprepared by reacting the corresponding3,3,2l-diacyloxy-l4-pregnen-20-one illusi8 trated by Examples 32-37 withhypobromous acid tHOBr) or hypochlorous acid the hypobromous acidpreferably prepared in situ.

EXAMPLE 44 313,21 -dihydr0xy-J 5 fi-hydroxy-J 4 u-br0m0pregnan-20-0ne Amixture of milligrams of3,8,2l-diacetoxy-l5flhydroxy-l4a-bromopregnan-20-one, five millilitersof methanol, one milliliter of water and 100 milligrams of sodiumcarbonate was heated for a period of thirty minutes, allowed to cool,and thereafter poured into twenty-five milliliters of water. Theprecipitate, an oily residue, was separated from the water layer,redissolved in acetone and twice recrystallized from acetone solution togive pure 35,21-dihydroxy-15/3-hydroxy-14a-br0- mopregnan-20-one.

EXAMPLE 45 3,8 acetoxy 15,3 hydroxy 14a bromo 14a card- 20(22)-en0lide Amixture of 1.55 grams of3,8,2l-diacetoxy-15fl-hydroxy-l4a-bromopregnan-20-one and 1.7 grams ofiodinated zinc granules was dried at 100 degrees for a period ofeighteen hours. The mixture was then introduced into fifty millilitersof dry dioxane. To this vigorously stirred mixture was then addeddropwise 2.55 grams of ethyl bromoacetate. The reaction mixture wascarefully regulated by cooling or warming as necessary. After thebromoacetate had been added the solution was warmed on a steam bath toeighty degrees centigrade for a period of thirty minutes. Thereafter thereaction mixture was cooled, diluted with fifty milliliters of dryethanol, filtered and mixed with ether and thirty milliliters of dilutehydrochloric acid. The aqueous layer was extracted twice more withether, the combined ether extracts were washed with Water, andconcentrated. The residue was purified by chromatographing over Florisilmagnesium silicate. Ethylene dichloride-acetone 3:1 eluted 855milligrams of 3,B-acetoxy-15/i-hydroxy-140tbromo-l4a-card-20(22)-enolideconfirmed. by infrared spectral data.

EXAMPLE 46 In the same manner as given in Example 45,Bit-propionlyoxy-Zl-acetoxy-l5,8-hydroxy-l4tx-bromopregnan 20- one wasreacted in solution with ethyl brlomoacetate to give the corresponding3,6-propionyloxy-15,8-hydroxy-14abromo-l4rx-card-20(22)-enolide.

EXAMPLE 47 Sfi-valeryloxy-I S S-hydrOxy-J 4a-br0mo-14ucard-20 (22-en0lide In the same manner as given in Example 45,3,8-valeryloxy-21-acetoxy-15,8-hydroxy-l4u-bromopregnan 20 one wastreated with ethyl bromoacetate in the presence of zinc to give3B-valeryloxy-1SB-hydroxy-14a-bromo-14acard-20(22)-enolide.

EXAMPLE 48 3 fi-acetoxy-J 5 ,B-hydroxy-I 4 Ot-Ch Zora-1 4occard-20(22)-enolide In the same manner as given in Example 45,3/3,2l-diacetoxy e hydroxy 141x chloropregnan-ZO-one was treated withethylbromoacetate in the presence of zinc to give3fl-acetoxy-l5;3-hydroxy-14u-chloro-l4a-card-20(22) enolide.

In the same manner as shown in Examples 45 through 48, other3B-acyloxy-lSflhydroxy-14a-halo-14a-card-20- (22)-enolides may beprepared by reacting the correspondin g 35,2 1 -diacyloxy- 15Bhydroxy-l4oc-ha1opregnan- 20-one with ethyl bromoacetate in thepresence of zinc. Representative 3 B acyloxy 15 ,8hydroxy-l4a-halo-14acard-20(22)-enolides thus obtained include:3fl-formyl oxy-, 3Bcyclop'entylpropionyloxy-, 3/3-isobutyryloxy-, 3B-isovaleryl'o'xyu 3B-heXanoyloxy-, 3fl-heptanoyloXy-, 3B- octanoyloxy-,3fl-(2-furoyloxy) 15,8-hydroxy-l-42x-br0mo- 14a-card-20(22)-enolide,'3B-formyloxy-, 3,8-propi'onyloxy-, 3B-butyryloXy-, 3(3-valeryloxyandZB-hexanoyloxylSfi-hydroxy-14a-chloro-14a-card-20(22)-enolide and thelike.

EXAMPLE 49 3fi-acet0xy-14U5 -xido-20(22 -cardenolide To asolution of0.95 gram of 3B-acetoxy-15fi-hydroxy-140t-bI'0Il'lO-14cL-C8Id-20(22)'CIlOlldB, dissolved in fifty millilitersof acetone was added 2.5 grams of potassium acetate. The reactionmixture was stirred at the tern perature of reflux in a dry atmospherefor a period of five hours. Thereafter the solution was concentrated atreduced pressure to a volume of ten milliliters and poured into 200milliliters of ice Water With stirring. The thusobtained precipitate wascollected on a filter and recrystallized from acetone to give 0.78 gramof 3B-acetoxy 14( 15) -oxido-20 22 cardenolide.

In the same manner as shown in Example 49, substituting3B-E1CetOXy-'1SB-hYdIOXY-14OL-bl0l'l'lO-14OL-C3l'd-20(22)- enolide with3,8-acetoxy-15 fi-hydroxy 14cc chloro 14acard-2O(22)-enolide produces3/3-acetoxy-14(15)-oxido- 20(22) -carde'nolide.

EXAMPLE 5O 3fl-propionyloxy-14U5 -oxido-20 (22 -carden0lide In the samemanner as shown in Example 49, 3B-Pl'OplOl'lY-IOXY-lSfl-hydIOXY-140t-bI'OmO-140L-C8Id 20(22)- enolide wastreated with soda lime to give 3ii-propionyloxy- 14 15 -oxido- 22-cardenolide.

EXAMPLE 5t 3/3-butyryloxy-14(15) -0xid0-20(22) -carden0lide In the samemanner as shown in Example 49,3,B-butyryloxy-lSfl-hydroxy-14u-bromo-14a-card 20(22) enolide wastreated with sodium acetate to give 3,8-butyryloxy-14(15)-oxido-20(22)-cardenolide.

In the same manner as shown in Examples 49 through 51, by reacting thecorresponding 3fl-acyloxy-l5fi-hydroxy-l4m-halo-14u-card 20(22)-enolide,illustrated by Examples 4548, with potassium acetate, sodium acetate,soda lime, and the like, the corresponding Zip-acyloxy- 14( 15)oXido-20(22) -cardenolide is obtained.

EXAMPLE 52 3;.8-hydr0xy-14 (15 -0xido-20(22 -carden0lide A medium wasprepared of ten grams of Cerelose dextrose, twenty grams of corn steepliquor and one liter of tap water. Twelveliters of this sterilizedmedium was inoculated with Rhizopus shanghaiensis ATCC 10329 andincubated for 24 hours at a temperature of 27 to thirty degreescentigrade, with agitation and aeration at a rate of one liter of airper minute. To this medium containing a twenty-four hour growth ofRhizopus shanghaiensis, 1.25 grams of 3B-acetoxy-l4(15)-oxido-20(22)-cardenolide, dissolved in 100 milliliters of acetone, was added.The incubation was continued for a period of four hours with agitationand without aeration. The beer and mycelium were extracted three timeswith three liters of methylene dichloride. The combined extracts werewashed with sodium carbonate solution and water. Upon concentration andtrituration of the extractives with ether 1.05 grams of3(3-hydroxy-14(15)-oxido-20(22)- cardenolide was obtained.

Other fungi of the genus Rhizopus, esterases or lipases may be used toproduce a rapid hydrolysis of the 3B- acyloxy group to a 3p-hydroxygroup without attacking the 20(22)-cardenolide lactone structure. Hog orbeef liver or pancreas are especially useful.

In a manner similar to Example 52, other3fl-acyloxy-14(15)-oxido20(22)-cardenolides such as those shown anddescribed in Examples 49 through 51, are

hydrolyzed to give BB-hydr'oXy-TMIS)-oxido-20(22)- cardenolide.

EXAMPLE 53 3 3,] 4-dihydroxy-20(22 )-carden0lide (digitoxigenin) Asolution of 500 milligrams of 3B hydroxy-14(15)-oxido-20(22)-cardenolide, dissolved'in 2'0milliliters of freshlydistilled dioxane was cooled to about ten degrees centrigrade. To thissolution was added thirty milligrams of potassium borohydride, and onemilligram of potassium chloride in five milliliters of fifty percentdioxane. The reaction mixture was then warmed to room temperature(twenty to thirty degrees centigrade) and kept for five hours at thistemperature. Thereafter five milliliters of water was added and a pH of2 obtained by adjustment with dilute .hydrochloric acid. After thirtyminutes the reaction mixture was concentrated in vacuo to one-fifty ofthe original volume. Dilution with twenty milliliters of water and;extraction with chloroform gave a residue of38,15-dihydroxy-20(22)-cardenolide. Separation was achieved. bychromatography over magnesium silicate. The component melting at 243 to247 degrees centigrade, was separated and identified by infraredspectrum, papergram mobility and melting point in admixture withauthentic digitoxigenin as 3 3,145- dihydroxy-20 (22) -cardeno1ide('digitoxigenin) EXAMPLE 54 Digitoxigenin acetate In the same manner asshown in Example 53, using 3fi-acetoxy-14( 15 -0XiC10-20(22)-carden0lide instead of 3B-hydroxy-l4(l5)-oxido-20(22)-cardenolideis productive of 3 8-acetoXy-14-hydroxy-20(20) -cardenolide(digitoxigenin-3 acetate).

EXAMPLE 55 3-( li-cyclopentylpropionyloxy)-14;8-hydraxy-20 (22cardenolide In the same manner as given in Example 53, using35-(fl-cyclopentyl)propionyloxy-14(15) oxido 20(22)- cardenolide insteadof 3/3-hydroxy-14(15)-oxido-2O'(22) cardenolide is productive of 3p-(flcyclopentyl)propionyloxy-14B-hydroxy-2O (22 -cardenolide (digitoxigenin3- )3- cyclopentyl)propionate).

In a manner similar to Examples 53 through 55, reducing3;.8-acylox'y-'14( 15 -oxidio-20(22)-cardenolide with potassiumborohydride or sodium borohydride, and the like, other3fi-acyloxy-i4(15)-oxido-20(22)-cardenolides are obtainable. Similarly 33-acyloxy-14{3-hydroxy-20- (22)-cardenolides may be obtained byesterifying 35,14,6-

dihydroxy-20(22)-cardenolide according to the methods as hereindescribed in pyridine solution with an acylating ing agent such as anacid halide, an acid anhydride, and the like, to obtain thecorresponding 35-acyloxy-14fl-hydroxy 20(22) cardenolide (digitoxigenin3 acylate). Representative 3fl-acyloxy-14fi-hydroXy'-20(22)cardenolidesthus-obtained comprise: f0rmyl0xy-, acetoxy-, propionyloxy-, butyryloxyisobutyry1oxy-, valeryloxy-, iso valeryloxy-, hexanoyloxy-,hep'tanoyloxy-, octanoyloxy-, benzoyloxy-, (,8cyclopentylpropionyloxy)-, dimethylacetoxy-, trimethylacetoxy-,phenylacetoxy-, toluyloxy-, anisoyloxy-, ga1lyloXy-,'salicyloyloxycinnamyloxyn hemisuccinyloxy-, hemitartaryloxy-,dihydrogencit'ryl- 0xy-, maleyloxy-, fumaryloxy-, crotonyloxy-,acryiyloxy (f3 methylcrotonyloxy)-, cyclohexanecarbonyloxy,chloroacetoxy-, dichloroaceto'xy, trichloroacetoXy-, bromoacetoxy,hemiquinolinoyloxy-, 'nicotinyloxy-, piper onyloxy-, (2-furoyloxy)-,thioglycollyloxy (para-chlorobenzoyloxy)-, (para-bromobenzoyloxy)-, v(meta-nitro benzoyloXy and 3 ,6- 3 ,S-dinitrobenzoyloxy llfl-hydroxy-ZO(22)-cardenolide, and the like.

It is to be understood that the invention is not to be limited to theexact detailsor exact compounds shown and described, as obviousmodificationsand equivalents will be apparent to one skilled in the art,and -theinven- 21 tion is therefore to be limited only by the scope ofthe appended claims.

We claim:

1. A process for the production of 3B,l4fi-dihydroxy- 20(22)-cardenolidewhich comprises: pyrolyzing 15o:- acyloxyprogesterone, wherein the acylradical is of a carboxylic acid containing from one to eight carbonatoms, inclusive, at a temperature between about 275 to about 400degrees centigrade to give 4,14-pregnadiene- 3,20-dione; subjecting4,l4-pregnadiene-3,ZO-dione to aerobic fermentation by Ophiobolusherpotrichus and recovering from the fermentation mixture2l-hydroxy-4,l4- pregnadiene-3,ZO-dione; hydrogenating 2l-hydroxy-4,l4-pregnadiene-3,20-dione with hydrogen in the presence of a palladiumcatalyst in a basic medium to obtain 21-hydroxy-l4-pregnene-3,2()-dione;acylating 2l-hydroxy-l4- pregnene-3,20-dione with an acylating agentselected from the group consisting of carboxylic acid anhydride andcarboxylic acid chlorides and bromides, wherein the carboxylic acidcontains from one to eight carbon atoms, inclusive, to obtain thecorresponding 21-acyloxy-l4-pregnone-3,20-dione; reducing the thusobtained 2lacyloxy l4-pregnene-3,20-dione with an alkali metalborohydride to give 2l-acyloxy-3a-hydroxy-l4-pregnen-20-one; esterifyingthe 3oi-hydroxy group of 2l-acyloxy-3u-hydroxygroup of 2l-acyloxy-3ahydroxy-l4-pregnen-20-one with an arylsulfonyl halide wherein the halidehas an atomic weight between 34 and 81, to obtain the corresponding 2l-acyloxy-B a-arylsulfonyloxy-14-pregnen-2 O-one; heating the21-acyloxy-3a-arylsulfonyloxy-l4-pregnen-20-one, dissolved in acarboxylic acid containing from one to eight carbon atoms, inclusive,and having a melting point below fifty degrees centigrade, with analkali-metal salt of the same acid to obtain the corresponding35,21-diacyloxy-l4-pregnen-20-one; treating the thus-obtained 318,21-diacyloxy-l4-pregnen-2-O-one with a hypohalous acid wherein the halogenis defined as above, to yield the corresponding 35,21 diacyloxy 15 8hydroxy 14a halopregnen-20-one; treating the thus-produced3,8,21-diacyloXy-l5 8-hydroxy-l-4a-halopregnen-20-one with ethylbromoacetate in the presence of zinc to produce the corresponding3,3-acyloxy-15,8-hydroxy-14a-halo-l4wcard-20 (22)-enolide; treating thethus obtained 3fl-acyloxy-l5fihydroxy-l4a-halo l4a-card-20(22)-enolidewith a base to produce 33 acyloxy-l4(l5)-oxido-20-(22)-cardenolide;hydrolyzing the thus obtained 3B-acyloxy-14(l5)-oxido-20(22)-cardenolide by means of fermentation under anaerobic conditionswith a fungus of the genus Rhizopus to obtain3,B-hydroxy-l4(15)-oxido-20(22)-cardenolide; and reducing the thusobtained 3fi-hydroxy-l4(l5)-oxido- 20(22)-cardenolide with an alkalimetal borohydride to obtain 3 8, 1 4B-dihydroxy-20 (22 -cardenolide.

2. A process for the production of 35,14,3-dihydroxy- 20(22)-cardenolidewhich comprises: pyrolyzing aacyloxyprogesterone, wherein the acylradical is of a carboxylic acid containing from one to eight carbonatoms, inclusive, at a temperature between 275 to 400 degrees centigradeto give 4,l4-pregnadiene-3,20-dione; subjecting4,l4-pregnadiene-3,20dione to aerobic fermentation by Ophz'obolusherpolrichus and recovering from the fermentation mixture2l-hydroxy-4,14-pregnadiene- 3,20-dione; hydrogenating21-hydroxy-4,14-pregnadiene with hydrogen in the presence of a palladiumcatalyst suspended on a zinc oxide-carbonate carrier to obtain21-hydroxy-14-pregnene 3,20 dione; acylating21-hydroxy-l4-pregnene-3,20-dione with an acylating agent selected fromcarboxylic acid anhydride and carboxylic acid chlorides and bromides,wherein the carboxylic acid contains from one to eight carbon atoms, toobtain the corresponding 2l-acyloxy-l4 pregnene-3,20-dione; reducing thethus obtained 2l-acyloxy-l4-pregnene-3,20-dione with sodium borohydrideto give 2l-acyloxy-3a-hydroxy- 14-pregnen-20-one; esterifying the3a-hydroxy group of 21-acyloxy-3a-hydroxy-l4-pregnen-20-one with abenzenesulfonyl chloride to obtain the corresponding 2l-acyloxy-Ba-benzenesulfonyloxy-14-pregnen-20-one; heating the thus obtained21-acyloxy 3a benzenesulfonyloxy-l-ipregnen-20-one, dissolved in acarboxy'lic acid containing from one to eight carbon atoms, inclusive,and having a melting point below fifty degrees centigrade, with analkali-metal salt of the same acid, to obtain the corresponding3,8,2l-diacyloxyl4-pregnen-20-one; treating the thus obtained35,2l-diacyloxyl4-pregnen-20-0ne with an N-halo hydrocarbon carboxylicacid amide in the presence of a mineral acid to yield the corresponding3,3,2l-diacyloxy-lSfi-hydroxy 14a halopregnan-ZO-one; treating the thusproduced 35,21-diacyloxy-lSfi-hydroxyl4az-halopregnan-20-one with ethylbromoacetate in the presence of zinc to produce the corresponding3,8acyloxyl5B-hydroxy-14whalo-l4a-card-20(22)-enolide; treating the thusobtained 3d-acyloXy-l5/3-hydroxy-l4a-halol4acard-20(22)-enolide withalkali metal acetate to produce 3B-acyloxy-14(l5)-oXidoZ0(22)-cardenolide; hydrolyzing the thus obtained3,6-acyloxy-l4(l5)-0xido-20(22)- cardenolide by means of fermentationunder anaerobic conditions with a fungus of the genus Rhizopus to obtain3fl-hydroxy-l4(l5)-oxido-20(22)-cardenolide and treating with an alkalimetal borohydride to obtain 35,14,6- dihydroXy-20(22)-cardenolide.

3. A process for the production of 3fi,l4 3-dihydroxy-20(22)-cardenolide which comprises: pyrolyzing 15aacetoxyprogesterone ata temperature between 275 to 400 degrees Centigrade to give4,l4-pregnadiene-3,20 dione; subjecting 4,l4-pregnadiene-3,20-dione toaerobic fermentation by Ophiobolus herpotrichus and recovering from thefermentation mixture 21-hydroxy-4,l4-pregnadiene- 3,20-dione;hydrogenating 21-hydroXy-4,l4-pregnadiene with hydrogen in the presenceof a palladium catalyst suspended on a zinc oxide-zinc carbonate carrierto obtain a 21-hydroxy-14-pregnene-3,20-dione; acylating with aceticanhydride and pyridine 2l-hydroxy-l4-pregnene- 3,20-dione to obtain2l-acetoxy-l4-pregnene-3,20-dione; reducing the thus obtained2l-acetoxy-l4-pregnene-3,20- dione with sodium borohydride to give2l-acetoxy-3ahydroxy-l4-pregnen-20-one; esterifying the 3a-hydroxy groupof 21-acetoxy-3whydroxy-14-pregnen-20-one with para-toluenesulfonylchloride to obtain 2l-acetoxy-3et-(para-toluenesulfonyloxy)-l4-pregnen-20-one; heating the2l-acetoxy-Su-(para-toluenesulfonyloxy)-14-pregnen 20- one, dissolved inglacial acetic acid, in the presence of sodium acetate, to obtain the35,2l-diacetoxy-l4-pregnen- 20-one; treating the thus obtained 33,2l-diacetoXy-l4- pregnen-ZO-one with N-bromoacetamide and perchloricacid to yield the corresponding313,21-diacetoxy-15fi-hydroxy-l4a-bromopregnen-20-one; treating the thusobtained 3,8,2l-diacetoxy-15B-hydroxy-14a bromopregnan- 20-one withethyl bromoacetate in the presence of iodinated zinc to produce3,8-acetoxy-l5fl-hydroxy-14a-bromol4a-card-20(22)-enolide; treating thethus obtained 3 3- acetoxy-15B-hydroxy-14a-bromo-l4a-card-20(22)-enolidewith potassium acetate to produce 3,8-acyloxy-l4(l5)-0xido-20(22)-cardenolide; hydrolyzing with esterase the thus obtained3,8-acetoxy-l4(l5)-oxido-20(22)-cardenolide to obtain3,8-hydroXy-l4(l5)-oxido-20(22)-cardenolide, and reducing the thusobtained 3,B-hydroXy-l4(l5)- oXido-20(22)-cardenolide with potassiumborohydride to obtain 3 B,l4,8-dihydroxy-20(22)-cardenolide.

4. A process comprising the hydrolysis of 3,8-acyloxy-14(15)-oXido-20(22)-cardenolide under anaerobic conditions by Rhizopusto obtain 3fi-hydroxy-14(l5)-oxido- 20(22)-cardenolide.

References Cited in the file of this patent UNITED STATES PATENTS Murrayet al. July 8, 1952 Wettstein et al. July 22, 1958 OTHER REFERENCES

1. A PROCESS FOR THE PRODUCTION OF 3B,14B-DIHYDROXY20(22)-CARDENOLIDEWHICH COMPRISES: PYROLYZING 15AACYLOXYPROGESTERONE, WHEREIN THE ACYLRADICAL IS OF A CARBOXYLIC ACID CONTAINING FROM ONE TO EIGHT CARBONATOMS, INCLUSIVE, AT A TEMPERATURE BETWEEN ABOUT 275 TO ABOUT 400DEGREES CENTIGRADE TO GIVE 4,14-PREGNADIENE3,20-DIONE, SUBJECTING4,14-PREGNADIENE-3,20-DIONE TO AEROBIC FERMENTATION BY OPHIOBOLUSHERPOTRICHUS AND RECOVERING FROM THE FERMENTATION MIXTURE21-HYDROXY-4,14PREGNADIENE-3,20-DIONE, HYDROGENATING21-HYDROXY-4,14PREGNADIENE-3,20-DIONE WITH HYDROGEN IN THE PRESENT OF APALLADIUM CATALYST IN A BASIC MEDIUM TO OBTAIN21-HYDROXY-14-PREGNENE-3,20-DIONE, ACYLATING21-HYDROXY-14PREGNENE-3,20-DIONE WITH AN ACYLATING AGENT SELECTED FROMTHE GROUP CONSISTING OF CARBOXYLIC ACID ANHYDRIDE AND CARBOXYLIC ACIDCHLORIDES AND BORMIDES, WHEREIN THE CAROXYLIC ACID CONTAINS FROM ONE TOEIGHT CARBON ATOMS, INCLUSIVE, TO OBTAIN THE CORRESPONDING21-ACYLOXY-14-PREGNENE-3,20-DIONE, REDUCING THE THUS OBTAINED21-ACYLOXY14-PREGNENE-3,20-DIONE WITH AN ALKALI METAL BOROHYDRIDE TOGIVE 21-ACYLOXY-3A-HYDROXY-14-PREGNEN-20-ONE, ESTERIFYING THE 3A-HYDROXYGROUP OF 21-ACYLOXY-3A-HYDROXYGROUP OF21-ACYLOXY-3A-HYDROXY-14-PREGNEN-20-ONE WITH AN ARYLSULFONYL HALIDEWHEREIN THE HALIDE HAS AN ATOMIC WEIGHT BETWEEN 34 AND 81, TO OBTAIN THECORRESPONDING 21-ACYLOXY-3A-ARYLSULFONYLOXY-14-PREGNEN-20-ONE, HEATINGTHE 21-ACYLOXY-3A-ARYLSULFONYLOXY-14-PREGNEN-20-ONE, DISSOLVED IN ACARBOXYLIC ACID CONTAINING FROM ONE TO EIGHT CARBON ATOMS, INCLUSIVE,AND HAVING A MELTING POINT BELOW FIFTY DEGREES CENTIGRADE, WITH ANALKALI-METAL SALT OF THE SAME ACID TO OBTAIN THE CORRESPONDING3B,21-DIACYLOXY-14-PREGNEN-20-ONE, TREATING THE THUS-OBTAINED3B,21DIACYLOXY-14-PREGNEN-20-ONE WITH A HYPOHALOUS ACID WHEREIN THEHALOGEN IS DEFINED AS ABOVE, TO YIELD THE CORRESPONDING 3B,21 -DIACYLOXY - 15B - HYDROXY - 14AHALOPREGNEN-20-ONE, TREATING THETHUS-PRODUCED 3B,21-DIACYLOXY-15B-HYDROXY-14A-HALOPREGNEN-20-ONE WITHETHYL BROMOACETATE IN THE PRESENCE OF ZINC TO PRODUCE THE CORRESPONDING3B-ACYLOXY-15B-HYDROXY-14A-HALO-14A-CARD-20 (22)-ENOLIDE, TREATING THETHUS OBTAINED 3B-ACYLOXY-15BHYDROXY-14A-HALO-14A-CARD-20 (22)-ENOLIDEWITH A BASE TO PRODUCE 3B - ACYLOXY-14 (15)-OXIDO-20(22)-CARDENOLIDE,HYDROLYZING THE THUS OBTAINED 3B-ACYLOXY-14 (15)-OXIDO20(22)-CARDENOLIDEBY MEANS OF FERMENTATION UNDER ANAEROBIC CONDITIONS WITH A FURGUS OF THEGENUS RHIZOPUS TO OBTAIN 3B-HYDROXY-14(15)-OXIDO-20(22)-CARDENOLIDE, ANDREDUCING THE THUS OBTAINED 3B-HYDROXY-14(15)-OXIDO20(22)-CARDENOLIDEWITH AN ALKALI METAL BOROHYDRIDE TO OBTAIN3B,14B-DIHYDROXY-20(22)-CARDENOLIDE.